Abstract

In plants, continuous formation of lateral roots (LRs) facilitates efficient exploration of the soil environment. Roots can maximize developmental capacity in variable environmental conditions through establishment of sites competent to form LRs. This LR prepattern is established by a periodic oscillation in gene expression near the root tip. The spatial distribution of competent (prebranch) sites results from the interplay between this periodic process and primary root growth; yet, much about this oscillatory process and the formation of prebranch sites remains unknown. We find that disruption of carotenoid biosynthesis results in seedlings with very few LRs. Carotenoids are further required for the output of the LR clock because inhibition of carotenoid synthesis also results in fewer sites competent to form LRs. Genetic analyses and a carotenoid cleavage inhibitor indicate that an apocarotenoid, distinct from abscisic acid or strigolactone, is specifically required for LR formation. Expression of a key carotenoid biosynthesis gene occurs in a spatially specific pattern along the root's axis, suggesting spatial regulation of carotenoid synthesis. These results indicate that developmental prepatterning of LRs requires an uncharacterized carotenoid-derived molecule. We propose that this molecule functions non-cell-autonomously in establishment of the LR prepattern.

Carotenoid-deficient seedlings produce few LRs. (A) Simplified version of the carotenoid biosynthesis pathway. Carotenoid- and apocarotenoid-specific biosynthesis steps are depicted in black, and upstream steps are shown in dark gray. Carotenoid biosynthesis mutants used in this study are indicated (gray italics), and those with an indirect impact are shown in parentheses. The dotted arrow to strigolactone represents unknown final steps. Columbia-0 (Col-0) seedlings were grown under control (B) and CPTA treatment (C) conditions. (D) Quantification of LR capacity in control and NF- or CPTA-treated seedlings. (E) Col-0 and ispH1/clb6, psy, and sca3 albino seedlings. (F) Quantification of LR capacity in Col-0 and albino seedlings. The difference between control and treated or control and mutant in D and F, respectively, is statistically significant (Student t test, P < 1 × 10−6). (G) CPTA-treated seedlings transferred to control medium (yellow dotted line indicates root tip position at transfer). Arrowheads indicate emerged LRs: those formed in the presence of CPTA (white) and those formed after transfer (yellow). Error bars represent SD. (Scale bars: 1 cm.)

Carotenoid-deficient plants have few prebranch sites. pDR5:LUC expression in control (A and B) and CPTA-treated seedlings (C and D). Luciferase activity (A and C) and overlays of luciferase activity and bright-field images (B and D) are shown. Color bars represent the range of analog-digital units (ADU) for luciferase activity. (Scale bars: 1 cm.) (E) Quantification of prebranch site number in control and CPTA-treated seedlings; the difference is statistically significant (Student t test, P < 1 × 10−6). Error bars represent SD. pDR5:LUC expression in the OZ is shown over time in control (F) and CPTA-treated (G) seedlings.